plants

Review
Amino Acid Transporters in Plant Cells:
A Brief Review
Guangzhe Yang *, Qiuxing Wei, Hao Huang and Jixing Xia *
State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science
and Technology, Guangxi University, Nanning 530005, China; weiqiux@163.com (Q.W.);
1908301017@st.gxu.edu.cn (H.H.)
* Correspondence: ygz201@163.com (G.Y.); xiajx@gxu.edu.cn (J.X.); Tel./Fax: +86-0771-3236616 (G.Y. & J.X.);




Received: 25 June 2020; Accepted: 28 July 2020; Published: 30 July 2020


Abstract: Amino acids are not only a nitrogen source that can be directly absorbed by plants, but also
the major transport form of organic nitrogen in plants. A large number of amino acid transporters
have been identified in different plant species. Despite belonging to different families, these amino
acid transporters usually exhibit some general features, such as broad expression pattern and substrate
selectivity. This review mainly focuses on transporters involved in amino acid uptake, phloem
loading and unloading, xylem-phloem transfer, import into seed and intracellular transport in
plants. We summarize the other physiological roles mediated by amino acid transporters, including
development regulation, abiotic stress tolerance and defense response. Finally, we discuss the
potential applications of amino acid transporters for crop genetic improvement.

Keywords: amino acids; transporter; uptake; phloem loading; phloem unloading; xylem-phloem transfer;
development regulation; stress tolerance; defense response; nitrogen use efficiency

1. Introduction
Nitrogen (N) is an essential macroelement for plant growth and development. Plants mainly
absorb inorganic N in the form of nitrate and ammonium from the soil, while organic N such as amino
acids, can also be taken up by plants [1]. The absorbed inorganic N can be assimilated into amino
acids in roots and/or leaves. Amino acids in roots are transported mainly to source leaves in the xylem
transpiration stream [2]. As the main transport form of organic N in most plant species, amino acids
synthesized in leaves or derived from roots are transported via phloem to developing sink organs to
meet their N requirement [3]. Therefore, the transport and allocation of amino acids within plants is
crucial for their growth, development and seed set.
Amino acids transport is generally mediated by transport proteins in plants. The first plant
amino acid transporters were cloned mainly through functional complementation of yeast mutants
deficient in amino acid transport [3–5]. Subsequently, amino acid transporters could be identified by
bioinformatics analysis after the completion of genome sequencing of some plants [6,7]. Now, the
presence of dozens, or even hundreds, of amino acid transporters are found in different plant species
(Table 1). These transporters belong to three major families: ATF (amino acid transporter family, also
called AAAP family), APC (amino acid-polyamine-choline transporter family) and the newly identified
UMAMIT (usually multiple acids move in and out transporter family) [8]. The ATF family generally
contains eight subfamilies: AAP (amino acid permeases), LHT (lysine and histidine transporters), ProT
(proline transporters), GAT (γ-aminobutyric acid transporters), ANT (aromatic and neutral amino
acid transporters), AUX (auxin transporters), ATL (amino acid transporter-like) and VAAT (vesicular
aminergic-associated transporters) [9]. The members of the AUX subfamily usually transport auxin
instead of amino acids [8,10]. The APC family consists of three subfamilies: CAT (cationic amino

Plants 2020, 9, 967; doi:10.3390/plants9080967 www.mdpi.com/journal/plants

Plants 2020, 9, 967 2 of 17

acid transporters), ACT (amino acid/choline transporters) and PHS (polyamine H+ -symporters) [11].
UMAMITs belong to the nodulin-like gene family [12], and recently several members of this family
have been reported to function as bidirectional amino acid transporters [13,14]. Among these families,
of main attention in plants is the AAP family. With respect to the phylogenetic relationship of these
transporters, readers are referred to other literature [7,15,16]. Despite belonging to different families,
these amino acid transporters display some common properties such as broad expression pattern
and substrate specificity. Here, we focus mainly on the amino acid transport processes and the
other physiological roles mediated by these transporters. The potential applications of amino acid
transporters for crop genetic improvement are also discussed in this review.

Table 1. Number of amino acid transporter family (ATF) and amino acid-polyamine-choline transporter
(APC) family members in some plant species.
ATF/AAAP a APC Others References
Species Total
AAP LHT ProT GAT ANT AUX ATL b VAAT b CAT ACT PHS TTP c
A. thaliana 63 8 10 3 2 4 4 5 10 9 1 5 2 [17,18]
rice 85 19 6 3 4 4 5 7 10 11 7 9 N. D. d [19]
soybean 189 35 24 7 19 6 16 16 30 19 7 9 1 [18]
poplar 100 17 13 3 7 4 8 8 11 16 6 7 N. D. [20]
maize 96 24 15 2 2 3 5 6 14 12 7 6 N. D. [20,21]
potato 72 8 11 4 3 5 5 8 8 9 1 8 2 [9]
wheat 283 60 24 9 12 18 15 55 e 31 19 31 9 [22]
R. communis 62 10 9 7 N. D. 15 f 4 15 2 N. D. N. D. [23]
M. truncatula 86 26 18 3 4 3 5 13 14 N. D. N. D. N. D. N. D. [24]
P. edulis 55 16 8 3 6 2 7 6 7 N. D. N. D. N. D. N. D. [25]
a : The full names of the abbreviations, ATF, APC, AAP, LHT, ProT, GAT, ANT, AUX, ATL, VAAT, CAT,

ACT and PHS, can be found in the main text. AAAP: amino acid/auxin permease; TTP: tyrosine-specific
transporter; A. thaliana: Arabidopsis thaliana; R. communis: Ricinus communis; M. truncatula: Medicago truncatula;
P. edulis: Phyllostachys edulis. b : The ATL and VAAT subfamilies are also known as ATLa and ATLb, respectively.
c : TTP, tyrosine-specific transporter, is classified in APC family in reference [9] but is classified in ATF family in

reference [22]. d : N.D. not determined. e : This contains the members of VAAT subfamily. f : This contains the
members of ATL and VAAT subfamily.

2. General Features of Plant Amino Acid Transporters

2.1. Broad Expression Pattern

Determining the gene expression pattern is crucial for studying the function of plant amino
acid transporters. Several technologies such as northern blot, reverse transcription-polymerase chain
reaction (RT-PCR) and the transcriptional GUS (β-glucuronidase) reporter assay have been employed to
detect the expression pattern of individual amino acid transporters. Recently, high throughput analyses
such as RNA sequencing have been used to simultaneously detect the expression of multiple amino acid
transporters [9,18,22]. Although the expression levels of individual amino acid transporter may differ,
most of them are ubiquitously expressed throughout the plant with relatively high abundance in certain
organs [26–29]. For example, StAAP1 exhibits high expression in mature leaves of potato, but weak
expression in roots, stems, sink tubers and sink leaves [26]. The transcript of AtANT1 was detected in
all Arabidopsis organs with highest abundance in flowers and cauline leaves [27]. In addition, the
transcript levels of some amino acid transporters change at different development stages or under
certain stress conditions, suggesting that their expression is developmentally or environmentally
regulated [19,22,24]. Overall, the broad expression patterns imply that each amino acid transporter
may process multiple physiological functions in planta. Of note, the well-studied AAP family genes
are found to be usually expressed in the vascular system of plants [29–31], suggesting that they play
important roles in the long-distance transport of amino acids.
With respect to their subcellular localization, most of the reported plant amino acid transporters
are located at the plasma membrane, while only a few of them are localized to the organelle membrane
(Figure 1B). For instance, the Arabidopsis GABA (γ-aminobutyric acid) transporter AtGABP (also
called BAT1 [32]), and cationic amino acid transporter AtCAT9 are localized on the mitochondrial
and vesicular membranes, respectively [33,34]. These organelle-localized transporters mediate the
intracellular translocation of amino acids between different compartments, which is important for their
Plants 2020, 9, 967 3 of 17

synthesis, conversion and storage [8,35]. Nevertheless, very few studies have been reported on such
Plants 2020, 9, x up to now, which will get more attention in future studies.
transporters 6 of 18

Figure 1. Overview of the function of amino acid transporters identified in plants. (A) Summary of
Figure 1. Overview of the function of amino acid transporters identified in plants. (A) Summary of
transporters involved in intercellular and long-distance transport of amino acids. Some genes that were
transporters involved in intercellular and long-distance transport of amino acids. Some genes that
proposed to participate in certain amino acid transport steps are marked with question marks due to
were proposed to participate in certain amino acid transport steps are marked with question marks
lack of direct evidence. (B) Summary of transporters involved in intracellular transport of amino acids.
due to lack of direct evidence. (B) Summary of transporters involved in intracellular transport of
Transporters with defined transport direction are marked with black arrows. References on these genes
amino acids. Transporters with defined transport direction are marked with black arrows. References
depicted in the figure can be found in the main text.
on these genes depicted in the figure can be found in the main text.
2.2. Broad Substrate Selectivity
3.2. Phloem Loading
The substrate specificity of plant amino acid transporters has been extensively studied in
plantaThe growth
[36] and inand development
heterologous of sink
systems organs
such depends
as yeast onand
[37,38] amino acidslaevis
Xenopus supplied by source
oocytes [39,40].leaves,
Most
of them are proton-amino acid symporters [3,4,41]. With the exception of the reported ProT and acids
and their phloem loading is a bottleneck in the source-to-sink translocation of N. The amino GAT
synthesized
family in mesophyll
members [28,42,43],cells can befamily
the other loaded into phloem
members, via a AAPs,
including symplastic
LHTs, or CATs
apoplastic transport
and ANTs, are
path, depending on the presence or absence of plasmodesmata between
usually capable of transporting a broad spectrum of amino acids with preference for some amino phloem parenchyma and
companion
acids cells, and their
[3,27,37,44,45]. frequencyOsAAP3
For example, [66]. Arabidopsis
can mediate andthe
most crops are
transport ofsuggested to be apoplastic
neutral, acidic and basic
phloemacids,
amino loaders
but[2], in which
transports thephloem loading
basic amino of amino
acids acidarginine
lysine and requiresrelatively
two key steps. Amino
well [40]. acids can
AtLHT2 are
firstly exported out of the phloem parenchyma cells of the minor veins into the
recognize amino acids with different charges but transports neutral amino acids with high efficiency [38]. apoplasm via passive
transport,
The and then including
ProT members, are pumped into the
AtProT1, phloem
AtProT2 andviaLeProT1,
active transport [2,67]. In this
seem to selectively pathway,
transport prolinethe
bidirectional
compared withamino acid transporter
the other proteinogenic SiAR1/AtUMAMIT18
amino acids [28,42].is This considered
substrateto preference
mediate amino mightacid be
export into the apoplasm [66,67] as it is expressed in both the major and
related to the demand for proline during plant development, since some ProTs such as LeProT1 are minor veins of source leaves
[13]. However,
specifically Besnard
expressed inetflowers
al. [54](especially
found that in loss-of-function of AtUMAMIT18
pollen) that accumulate does proline
much more not change amino
compared
acid composition in leaf phloem exudates. Hence, further research
with vegetative tissues [42]. Very few GAT members have been reported so far, and AtGAT1 fromis needed to determine whether
AtUMAMIT18
Arabidopsis plays a role
is described as in aminoselective,
a highly acid export from phloem
high-affinity GABAparenchyma
transporter cells, or which facilitator
[43].
compensates for theacids,
Besides amino loss ofother
AtUMAMIT18
substrates can function.
also be transported by some amino acid transporters.
Several amino acid importers such as
These substrates are mainly amino acid-related compounds, AtAAP2 [30], AtAAP8
including [68]indole-3-acetic
and AtProT1 acid [46] (IAA)
have been
[27],
shown to
glycine be expressed
betaine [42,46],in the leaf[42],
choline phloem, but only AtAAP8 is determined
1-aminocyclopropane-1-carboxylic to function
acid in amino
(ACC) [47,48], acid
amino
phloem loading from leaf cells so far [68]. When 14C-labeled glutamate or glutamine was fed to source
leaves, the label transported to sink leaves and siliques decreased significantly in ataap8 mutants. In
addition, the total amino acid concentrations were also markedly reduced in the ataap8 leaf exudates
[68]. Consequently, decreased phloem loading and source-to-sink transport of amino acids resulted
in reduction of silique and seed numbers, and yield in ataap8 mutants [68]. However, since the phloem
Plants 2020, 9, 967 4 of 17

acid-based pesticides [49–51] and so forth. For example, two members of LHT family, AtLHT1
and AtLHT2, have been reported to be able to transport the biosynthetic precursor of ethylene,
ACC, which is also a non-proteinogenic α-amino acid [47,48]. Disruption of AtLHT1 impairs the
exogenous ACC-induced ethylene responses, while overexpression of AtLHT1 or AtLHT2 can restore
the ACC-resistance phenotype of atlht1 mutants [47,48]. More recently, several studies have shown that
AtLHT1 can also mediate the translocation of amino acid-pesticide conjugates within plants [49,50].
Although ProT proteins fail to transport efficiently proteinogenic amino acids other than proline, they
exhibit higher affinity for glycine betaine than for proline [42,46], so they may be involved in the
transport of these compatible solutes in plants. The multiplicity of substrates of some amino acid
transporters also suggests that they may play various physiological roles in planta.

2.3. Multiple Physiological Functions of Some Amino Acid Transporters

The in planta functions of amino acid transporters have been extensively studied with genetic,
physiological and biochemical methods in the past two decades. They participate in multiple amino
acid transport processes within plants, including amino acid intracellular and intercellular translocation,
uptake from medium, phloem loading and unloading, xylem-phloem transfer and post-phloem delivery
into sink cells (Figure 1; see below). It is worth noting that several amino acid transporters have been
reported to have more than one physiological role in planta [14,37,52–54]. For example, initial studies
demonstrated a function of AtAAP1 in root amino acid uptake from medium [52]. Subsequently,
Sanders et al. [53] found that AtAAP1 also mediates the import of amino acids into developing embryos.
AtLHT1 was shown to participate in amino acid import in roots as well as in mesophyll cells, so its
knockout mutants displayed retarded growth when grown on aspartate or glutamate as the sole N
source, and decreased amino acid uptake in mesophyll protoplasts [37]. Similarly, AtUMAMIT14
is involved in phloem unloading of amino acids in root and seed [14,54]. The pleiotropy of these
transporters poses challenges in studying their function and interpreting obtained results. Thus, it is
necessary to keep this pleiotropy in mind when performing experiments to dissect their functions.
For example, to exclude the possible interference of AtAAP1 function in root amino acid uptake with
its role in the seed, the researchers only supplied amino acid-free N fertilizer to plants in the study [53].
On the other hand, these amino acid transporters may have overlapping, at least partially overlapping,
functions within plants, as indicated by no visible phenotypic changes observed in some of their
knockout mutants [55–57].

3. Amino Acid Transport Processes Mediated by Amino Acid Transporters

3.1. Acquisition of Amino Acids by Roots

Several amino acid transporters, including AtLHT1 [37], AtLHT6 [58], OsLHT1 [59], AtAAP1 [52],
AtAAP5 [60] and AtProT2 [61], have been reported to be able to take up amino acids from external
medium (Figure 1A). These findings are mainly based on the observations that mutants defective in
certain amino acid transporters show growth retardation on medium containing amino acid as the sole
N source [37], reduced uptake of amino acids [36,58] or better growth (survival) on toxic levels of some
amino acids or their analogues [52,58]. The uptake characteristics of these transporters generally differ
in amino acid type and concentrations. AtLHT1 is involved in uptake of glutamine, alanine, glutamate
and aspartate at low concentrations, but not arginine or lysine, while AtAAP5 can mediate the uptake
of arginine and lysine under the same condition [36]. Unlike AtLHT1 and AtAAP5, AtAAP1 may
function in amino acid acquisition only at high concentrations, as its mutation does not change the
uptake of any of the tested amino acids at low concentrations [36].
It is noteworthy that experiments determining the uptake function of plant amino acid transporters
are generally performed in agar medium or nutrient solutions [36,37,52,58] which contain enough
free amino acids and are usually under appropriate pH and sterile conditions. However, the soil
environment for growing crops is much more complex than these simulated environments, such as
Plants 2020, 9, 967 5 of 17

having very low concentrations of free amino acids [62,63], inappropriate (neutral or alkaline) pH
or strong competition for amino acids with large amounts of soil microorganisms [64,65]. Therefore,
it remains to be determined whether these transporters are capable of taking up amino acids from soils,
and how much they contribute to plant N acquisition in a cropland system.

3.2. Phloem Loading

The growth and development of sink organs depends on amino acids supplied by source leaves,
and their phloem loading is a bottleneck in the source-to-sink translocation of N. The amino acids
synthesized in mesophyll cells can be loaded into phloem via a symplastic or apoplastic transport
path, depending on the presence or absence of plasmodesmata between phloem parenchyma and
companion cells, and their frequency [66]. Arabidopsis and most crops are suggested to be apoplastic
phloem loaders [2], in which phloem loading of amino acid requires two key steps. Amino acids are
firstly exported out of the phloem parenchyma cells of the minor veins into the apoplasm via passive
transport, and then are pumped into the phloem via active transport [2,67]. In this pathway, the
bidirectional amino acid transporter SiAR1/AtUMAMIT18 is considered to mediate amino acid export
into the apoplasm [66,67] as it is expressed in both the major and minor veins of source leaves [13].
However, Besnard et al. [54] found that loss-of-function of AtUMAMIT18 does not change amino
acid composition in leaf phloem exudates. Hence, further research is needed to determine whether
AtUMAMIT18 plays a role in amino acid export from phloem parenchyma cells, or which facilitator
compensates for the loss of AtUMAMIT18 function.
Several amino acid importers such as AtAAP2 [30], AtAAP8 [68] and AtProT1 [46] have been
shown to be expressed in the leaf phloem, but only AtAAP8 is determined to function in amino
acid phloem loading from leaf cells so far [68]. When 14 C-labeled glutamate or glutamine was fed
to source leaves, the label transported to sink leaves and siliques decreased significantly in ataap8
mutants. In addition, the total amino acid concentrations were also markedly reduced in the ataap8
leaf exudates [68]. Consequently, decreased phloem loading and source-to-sink transport of amino
acids resulted in reduction of silique and seed numbers, and yield in ataap8 mutants [68]. However,
since the phloem loading and source-to-sink transport of amino acids are not completely aborted in
ataap8 plants, other unidentified transporters with overlapping function with AtAAP8 may exist in
Arabidopsis, which needs to be further explored in future studies.

3.3. Xylem-Phloem Transfer

Root-derived amino acids are transported to the shoot with the xylem transpiration stream. Along
the long-distance translocation pathway, some amino acids may move from the xylem to the phloem for
direct N supply to sink tissues [2]. This transfer process requires several steps: first, amino acids move
from tracheary elements into xylem parenchyma cells, and then move to phloem parenchyma cells
via plasmodesmata. Due to the lack of plasmodesmata between phloem parenchyma and companion
cells, the amino acids are further exported into the apoplasm followed by import into the sieve
element-companion cell (SE-CC) complexes [66]. AtAAP2 and AtAAP6 are thought to function in
xylem-phloem transfer of amino acids [30,31]. Owing to the low amino acid concentrations in xylem
sap, high-affinity transporters are required to mediate the import of amino acids into xylem parenchyma
cells. AtAAP6 can transport acidic and neutral amino acids with low Km values [39], and is localized
to the xylem parenchyma cells, so it is proposed to function in the import of amino acids diffusing out
of the tracheary elements into xylem parenchyma [31]. However, subsequent studies showed that loss
of function of AtAAP6 results in marked reduction of total amino acid concentrations in sieve element
sap [69]. This result cannot be caused by the reduction in the xylem-phloem transfer of amino acids, as
concentrations of amino acids in the xylem are much lower than in the phloem sap [70,71]. Even if the
xylem-phloem transfer of amino acids is entirely aborted, the levels of amino acids in phloem sap does
not significantly decline. Therefore, additional evidence is required to determine the role of AtAAP6
Plants 2020, 9, 967 6 of 17

in xylem-phloem transfer of amino acids. Alternatively, AtAAP6 may have other unknown roles in
amino acid transport, which can regulate phloem amino acid concentration in Arabidopsis.
Unlike AtAAP6, unambiguous evidence supported that AtAAP2 functions in xylem-phloem
transfer of amino acids: (a) AtAAP2 is localized in the phloem and particularly in companion cells [30];
(b) total free amino acid levels in the xylem sap of ataap2 mutants are slightly higher than those in wild
type [30]; (c) when roots were fed with 14 C-labeled glutamine, ataap2 mutants display a significant
increase of 14 C-label in source leaves versus the wild-type, but a decrease in sink leaves and siliques [30].
These results indicate that knockout of AtAAP2 leads to reduction in xylem-phloem transfer of amino
acids, thereby increasing the transport of root-derived amino acids to mature leaves, while reducing
their transport to sinks via phloem. Unexpectedly, enhanced amino acids allocation to leaves has
positive effects on ataap2 growth, yield and nitrogen use efficiency (see below).

3.4. Phloem Unloading

Depending on plant species and sink organs, phloem unloading of assimilates may follow a
symplastic or apoplastic path. In roots, sink leaves and seeds the unloading mechanism of amino acids is
generally thought to be symplastic [10,67], so the exporter responsible for amino acid phloem unloading
has not been found for a long time. Recently, the functional characterization of UMAMIT efflux systems
has changed the understanding of phloem unloading of amino acids in these organs. Some UMAMITs
can facilitate import and export of amino acids with broad substrate specificity [13,14]. In seeds,
AtUMAMIT11, AtUMAMIT14 and AtUMAMIT18 are localized in the nutrient-unloading domain, so
they may mediate amino acid release from the phloem at the end of the funiculus vasculature [13,14].
AtUMAMIT14 is also expressed in root pericycle and phloem, and its knockout mutants exhibit
decreased shoot-to-root and root-to-medium transport of leaf-fed amino acids, so AtUMAMIT14 is
suggested to function in phloem unloading of amino acids in root as well [54].

3.5. Post Phloem Transport

Amino acids released from phloem move to terminal sink cells via symplastic and apoplastic
routes, depending on plant species, sink tissues and developmental phase [67]. In roots and sink
leaves, post-phloem translocation of amino acids occurs symplastically [67]. In endospermic seeds,
however, the three constituting tissues (seed coat, endosperm and embryo) are symplastically isolated
from each other, so amino acids need to be exported and subsequently reimported at least two times in
these seeds for final uptake by the embryo [66,67]. Consistent with these complex transport pathways,
many amino acid importers and exporters have been found to be expressed in seed (Figure 1A).
The importers include AtAAP1 [53], AtAAP8 [31] and AtCAT6 [45]. AtAAP1 is expressed in the
storage parenchyma and outer epidermis cells of the developing embryo [53]. The total carbon and
N amounts are significantly decreased in ataap1 seeds, whereas the content of total free amino acids
are elevated in ataap1 seed coat/endosperm, suggesting that knockout of AtAAP1 reduces the uptake
of amino acids by the embryo, thereby leading to an accumulation of free amino acids outside the
embryo [53]. AtAAP8 is expressed in young seeds [31], and its deletion leads to strong reduction in
seed number [72]. Thus, AtAAP8 may be involved in import of amino acids into developing seeds.
Except for glutamine, however, no difference in individual amino acid content was detected between
ataap8 and wild-type seeds [72]. Therefore, the exact role of AtAAP8 in amino acid transfer in the seed
remains to be resolved. Recently, AtAAP8 was found to function in phloem loading of amino acids
in source leaves [68], so the seed phenotype of ataap8 mutants might also be caused by reduction of
source-to-sink transfer of amino acids. Similarly, despite expression in the developing seed, knockout
of AtCAT6 did not change amino acid content in the seed, so the function of AtCAT6 in the seed
requires further investigation [45].
Several UMAMIT members expressed in the seed fulfill the function of amino acid export
(Figure 1A). These UMAMIT genes are expressed in distinct tissues within developing seeds and play
different roles in amino acid transfer from the mother to daughter tissues (for a review see [8,10]).
Plants 2020, 9, 967 7 of 17

For example, AtUMAMIT29 is localized in the middle layer of the inner integument and it may mediate
amino acid export from the outer integument to the inner integument [14]. Repression of its expression
has no impact on seed set, but seed volume is significantly decreased [14]. The tonoplast-localized
AtUMAMIT24 was found mainly in the chalazal seed coat and may be involved in temporary storage
of amino acids in chalaza [73]. AtUMAMIT25 is targeted to endosperm cells, indicating its role in
amino acid export from the endosperm [73].

3.6. Intracellular Translocation of Amino Acids

Amino acid metabolism is compartmented, so their transport between different organelles and
cytoplasm occurs frequently. While numerous plasma membrane-localized amino acid transporters
have been reported, very few organelle-localized amino acid transporters have been functionally
identified so far (Figure 1B). The Petunia hybrida cationic amino acid transporter, PhpCAT, is one of the
well-studied transporters localized to organelles [74]. Phenylalanine (Phe) is mainly synthesized in
plastids, while the synthesis of Phe-derived compounds is a complex multi-compartmental process.
Hence, Phe must be first exported to the cytosol across the plastid membrane. PhpCAT, localized to the
plastid, was shown to be responsible for plastidial Phe export [74]. Repression of PhpCAT expression
reduces Phe, tyrosine and tryptophan (to a lesser extent) levels in cytosol, as well as the total emission of
Phe-derived volatiles. In contrast, overexpression of PhpCAT results in increased levels of Phe-derived
volatiles and aromatic amino acid pools in the cytosol [74]. GABA is synthesized in cytosol but is
catabolized to other metabolites in mitochondria. AtGABP is suggested to function as a mitochondrial
GABA transporter mediating the import of GABA from the cytosol into mitochondria [33].
The vacuole can serve as a large amino acid pool in plant cells [75]. Amino acids import into
and export out of the vacuole requires the activity of transporters. Several amino acid transporters
belonging to different families have been found to be localized on the tonoplast [56,73,76]. AtAVT3
family members are homologs of AtANT1 but localized to the vacuolar membrane in Arabidopsis [76].
When expressed in a yeast mutant defective in amino acid export from vacuoles, they can reduce the
accumulation of amino acids within the vacuoles. Thus, AtAVT3 family members are proposed to
function as vacuolar amino acid exporters in Arabidopsis [76]. Some CAT family members such as
AtCAT2 and AtCAT4 were also found to be localized to the tonoplast [56,77]. Knockout of AtCAT2
significantly increases the total soluble amino acid content in adult leaves, suggesting the involvement
of AtCAT2 in regulation of leaf amino acid levels [56]. Nevertheless, it is unclear that the increase
in amino acids is caused by their accumulation in cytosol or vacuoles, so the transport direction of
AtCAT2 remains uncertain.

4. Other Physiological Roles of Plant Amino Acid Transporters

4.1. Regulation of Plant Development

Altered expression of amino acid transporters can influence internal amino acid homeostasis in
plants, ultimately leading to changes in plant metabolism, growth and development. However, these
changes may be caused by different factors: first, alterations in plant growth and development may be
due to the insufficiency of amino acids as a N source for sinks, which has been outlined in Section 3.
On the other hand, besides serving as fundamental nutrients, amino acids can also act as regulators to
modulate plant growth and development, which will be summarized in this section.
High concentrations of amino acids inhibit growth in both monocots and dicots [29,52,57,78].
In rice, low levels of amino acids can promote tiller bud outgrowth and tiller formation, while
their excessive accumulation retards tiller bud outgrowth and reduces tiller number [29,78]. Altered
expression of some amino acid transporters can regulate rice tillering by changing internal amino acid
homeostasis [29,78]. The rice amino acid permease 5, OsAAP5, is expressed in various organs and may
mediate the transport of basic and neutral amino acids in plants. Its expression level differs between
indica and japonica rice varieties. Indica cultivars with low expression of OsAAP5 produce more
Plants 2020, 9, 967 8 of 17

tillers than japonica cultivars, and vice versa [29]. Reduced expression of OsAAP5 in japonica varieties
significantly reduces the concentrations of some basic and neutral amino acids in the tiller basal part,
leaf sheath and leaf blade, whereas tiller number increases. Correspondingly, the opposite results
are observed in OsAAP5-overexpressing plants [29]. Hormones play important roles in regulating
plant development. The levels of cytokinins (CKs) are changed in the modified plants, so OsAAP5
regulates rice tiller formation probably by affecting CK levels in plant cells [29]. Nevertheless, it is
still obscure how amino acid accumulation reduces CK content in rice. Additionally, manipulation
of another OsAAPs gene, OsAAP3, produces a similar effect on rice development as OsAAP5 [78].
Unlike these two OsAAP genes, altered expression of another type of amino acid transporter, OsLHT1,
has the opposite effect on rice tillering. Knockout of OsLHT1 reduces rice tiller number and shoot
biomass [59,79], suggesting that OsLHT1 and OsAAP3/OsAAP5 might play different roles in transport
and allocation of amino acids in rice.
In Arabidopsis, overexpression of AtCAT1 leads to shoot growth inhibition, earlier flowering and
senescence [57]. However, only minor differences in the amino acid profile were found in adult leaves
of AtCAT1-OE and wild-type plants, whereas the expression of genes involved in salicylic acid (SA)
biosynthesis and SA levels are elevated in the transgenic plants, indicating the involvement of SA in
the developmental alterations of AtCAT1-OE plants [57]. From these examples, it can be assumed
that altered expression of amino acid transporters regulates plant development, probably by affecting
hormone action.

4.2. Abiotic Stress Tolerance

Proline is an important compatible solute in plants, and its accumulation can protect plants from
abiotic stresses such as salinity, drought and freezing. Besides enhanced synthesis and decreased
degradation, transport of proline within plants may also contribute to its accumulation [80,81] as
exemplified by the observation that the proline concentration is sharply increased in the phloem sap
of water-stressed alfalfa [82]. Several different types of amino acid transporters, including ProTs,
AAPs and LHTs, have been reported to have the activity of transporting proline [37,39,46], and altered
expression of some of these transporters can enhance plant stress tolerance [83,84]. In soybean, two ProT
genes, GmProT1 and GmProT2, are strongly induced by salt, drought and ABA treatment. Arabidopsis
plants over-expressing GmProT1 or GmProT2 accumulate more proline relative to wild-type under salt
and drought stresses and enhance the expression of stress-related genes. As a result, the overexpressor
lines show improved salt and drought tolerance [83]. GABA, another stress-induced compound in
plants, can function as an endogenous signaling molecule that regulates plant growth, development
and stress responses [85]. When the GABA transporter PeuGAT3 was overexpressed in Arabidopsis
and poplar, the transgenic plants showed increased thickness of xylem cells walls. Furthermore, the
roots of transgenic Arabidopsis grow much better than the wild type under salt and drought stresses,
suggesting that overexpression of PeuGAT3 enhances abiotic stress tolerance in Arabidopsis [86].

4.3. Defense Response

Plants are frequently infected by diverse pathogens and pests in the natural environment.
Upon infection, invaders acquire nutrition from their hosts and amino acids are an important source
of N provided by host plants [87]. Therefore, regulating amino acid homeostasis through changing
the expression of transporters might influence the plant defense response. AtLHT1 participates in
the uptake of amino acids into roots and mesophyll cells [37]. Liu et al. [88] demonstrated that the
expression of AtLHT1 is induced by pathogen infection. Its knockout mutation increases resistance to
a broad spectrum of pathogens in an SA-dependent fashion, evidenced by the observation that the
induction of AtLHT1 by pathogen infection and atlht1-conferred resistance is diminished in mutants
defective in SA pathway. The enhanced disease resistance in atlht1may be due to alteration in cellular
redox homeostasis and deficiency of cytosolic glutamine [88]. Unlike AtLHT1, overexpression of
AtCAT1 enhances resistance of Arabidopsis to bacterial pathogens [57]. As in the atlht1 mutant, the
Plants 2020, 9, 967 9 of 17

expression of the SA-synthesis genes ICS and endogenous SA amount are significantly elevated in
AtCAT1-OE plants, suggesting that increased pathogen resistance of the modified plants may also
depend on the SA pathway [57]. Nevertheless, the exact link between the altered expression of amino
acid transporter and the accumulation of SA is still obscure.
Altered expression of amino acid transporters can also influence the resistance to plant-parasitic
pests, such as root-knot and cyst nematodes [89–91]. For example, knockout of AtAAP3 and AtAAP6
significantly diminished the root-knot nematode infestation levels in Arabidopsis. Both ataap3 and
ataap6 mutants produced fewer female nematodes, but more males in comparison to wild-type plants.
Moreover, nematodes isolated from ataap3 and ataap6 mutants exhibit reduced egg hatching, infectivity
and lipid energy reserves [89]. Similarly, loss of function of AtAAP1, AtAAP2 or AtAAP8 markedly
reduced the number of female cyst nematodes propagated on these mutant plants [90].

5. Potential Applications of Amino Acid Transporters for Crop Improvement

5.1. Promoting Plant Growth and Increasing Yields

As the dominant transport form of organic N in plants, the transport and allocation of amino acids
is crucial for plant growth and development. Altered expression of amino acid transporters could affect
not only plant N metabolism, but also carbon (C) metabolism due to their strong interaction [92,93].
Dependent on the transporters, their overexpression (OE) or knockout may improve plant growth
and seed yield. For example, additional copies of PsAAP1 were overexpressed in pea under control of
the AtAAP1 promoter, which targets expression in the phloem and the cotyledon transfer cells [94].
In PsAAP1-OE plants, phloem loading and embryo loading of amino acids, as well as root N uptake
and assimilation, and root-to-shoot amino acid delivery are increased. As a result, the PsAAP1
overexpressors show increased shoot biomass and pod and seed number. Ultimately, the seed yield
per plant can be enhanced by up to 33% in the transgenic plants [94]. Moreover, the total seed protein
amount per plant is also elevated [94].
Different from PsAAP1, down-regulation of some amino acid transporters has positive influence
on plant growth and productivity [29,30,78]. As mentioned above, repression of the expression of
OsAAP5 or OsAAP3 increases rice tiller number and grain number per plant, ultimately leading to a
significant improvement in rice grain yield [29,78]. These valuable genes can be applied in rice breeding
programs and may contribute to cultivating high-yield rice cultivars in the future. In Arabidopsis,
loss-of-function of AtAAP2 reduces the source-sink translocation of amino acids and thus more N is
allocated to the mutant leaves. Increase in the leaf N supply leads to higher rubisco levels, chlorophyll
content, photosynthetic rates and C assimilate export from leaves for sink C supply in ataap2 plants.
Consequently, the branch and silique number per plant are elevated to different extents in ataap2 plants,
ultimately resulting in an increase in total seed and oil yield [30,95]. This study indicates that decrease
in source-to-sink transport of amino acids does not necessarily reduce seed yield, whereas it represents
an alternative strategy to boost crop productivity.

5.2. Seed/Fruit Quality Improvement

About 65% of the global edible protein supplied for human nutrition is derived from plants [96].
Hence, the contents of protein and amino acids are key factors determining the nutritional quality of
seeds. Generally, all proteinaceous amino acids are transported through phloem from source leaves
to seeds, and then are used for synthesis of proteins. Altering amino acid transport via regulating
transporters can ultimately affect the amino acid and protein content of seeds [26,97–101]. Rice is
generally thought of as a cereal with lower grain protein content (GPC) [102], while significant
genetic variations in GPC exist among different rice varieties [103]. Peng et al. [99] isolated a gene
controlling GPC in the rice grain by a map-based cloning strategy, which encodes a putative amino
acid transporter OsAAP6. Its transcripts are ubiquitously expressed in a variety of organs, but most
abundant in the developing endosperms. When the coding region of OsAAP6 from the high-GPC
Plants 2020, 9, 967 10 of 17

variety was overexpressed in the low-GPC variety, the engineered plants increase GPC. In contrast,
RNAi-mediated knockdown of OsAAP6 reduces GPC in the high-GPC variety [99]. Importantly,
alteration in OsAAP6 expression has no significant impact on other agronomic traits such as grain yield.
Therefore, manipulating OsAAP6 represents a valid strategy for enhancing GPC in rice and, potentially,
other gramineous crops [99]. In the legumes pea and Vicia narbonensis, when the Vicia faba amino acid
permease VfAAP1 was specifically expressed in their embryos, transgenic seeds had higher amino
acid import rates compared with the wild type, ultimately leading to elevated seed N and protein
content [100].
In addition to increased amino acid and protein content in seeds, the palatability or quality of
fruits can also be improved via genetic manipulation of amino acid transporters. The content of acidic
metabolites is an important factor in determining tomato fruit quality. As the fruit ripens, the acidic
amino acids aspartate and glutamate accumulate in the vacuole, while GABA level declines [104].
The tonoplast-localized transporter SlCAT9 was found to mediate the export of GABA from the vacuole
in exchange for the import of aspartate or glutamate. Its overexpression resulted in many-fold increases
in the contents of all three amino acids in ripe fruit [105]. Thus, amino acid transporters can also serve
as valuable engineering targets for improving fruit flavor and nutritional quality.

5.3. Enhancing Plant Nitrogen Use Efficiency

In agricultural production, N fertilizers are usually applied to obtain a high yield, but crops
take up only 30–40% of the applied N fertilizer [106]. The remaining soil N may be lost through
surface runoff, leaching, denitrification and volatilization, which cause serious environmental pollution.
To reduce production costs and potential environmental risks, increasing the nitrogen use efficiency
(NUE) of crops is an urgent issue in agricultural production. NUE is defined as the total biomass or
grain yield produced per unit of applied fertilizer N, which is generally composed of two physiological
components: nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE) [107].
NUpE is defined as the total N in plants relative to the applied N fertilizer, reflecting the capacity of
plants to acquire N from the soil, while NUtE is defined as plant biomass or seed yield relative to
total N in the plant, reflecting the capacity of plants to convert acquired N to plant biomass or seed
yield [107]. Nowadays, many genes involved in inorganic N uptake, allocation and assimilation, as
well as their regulation, have been used as genetic engineering targets for improving plant NUE [2,108].
However, taken into account that (a) as the main transport forms of organic N in most plants, large
amounts of amino acids are required for transport via vasculature to the sink organs, supporting their
growth, development and fruit/seed set, and (b) N is a highly mobile element. Proteins are degraded
in older and senescing leaves, and then transported mainly in the form of amino acids and peptides to
developing organs for reuse [109], manipulating amino acid transporters can also promote N allocation
and reutilization within plants, eventually increasing NUE and seed yield.
Indeed, improved NUE has been reported in different plant species by altering the expression
of amino acid transporters [78,95,110,111]. For example, pea plants overexpressing PsAAP1 in the
leaf phloem and embryos exhibited increased source-to-sink translocation of amino acids, seed yield,
as well as NUE under low, moderate and high N environments [110]. PsAAP1-OE plants display
improved NUtE under low and moderate N supply, while no difference in NUtE was found in a high
N condition. Unlike NUtE, NUpE was unaltered in PsAAP1 overexpressors under low N conditions
but was enhanced when moderate or high N was supplied [110]. More importantly, PsAAP1-OE plants
grown under moderate N condition produce similar yields to the wild type supplied with high N
(twice the moderate N) [110]. These results indicate that modulating source-to-sink translocation of N
could be a feasible strategy for improving crop NUE.
Different from PsAAP1, disruption of AtAAP2 leads to enhanced NUE in Arabidopsis plants in a
range of N environments [95]. Detailed analyses showed that the increase in NUE is caused by the
higher NUpE in ataap2 knockout plants, while the NUtE is unchanged [95]. The lack of difference in
NUtE between ataap2 and wild type is probably because loss of function of AtAAP2 reduces N transfer
Plants 2020, 9, 967 11 of 17

from vegetative organs to seeds, as indicated by observations that more N is retained in ataap2 stubble
tissues at harvest, while ataap2 seed N content is reduced [95].
Unexpectedly, both PsAAP1 and AtAAP2 are involved in transport and partitioning of amino
acids in the shoot, but alterations in their expression simultaneously enhance root N uptake and
assimilation, as well as root-to-shoot N delivery [94,95,110]. The reasons for this effect are very complex.
First, changes in shoot N concentrations or status probably influence shoot-to-root signaling and trigger
positive feedback regulation of root N uptake and assimilation [94,95]. Alternatively, alterations in
shoot N metabolism affect plant C metabolism (probably increased photosynthesis and sugar delivery),
and C assimilates provide the energy and C skeleton for root N uptake and assimilation [94,95].
It should be noted that the increased plant yield and NUE presented in these studies is obtained
usually from pot experiments and under controllable environments. Whether the good performances
of these genetically modified plants can be achieved in farmland is still to be tested. But anyway,
altering plant N transport and allocation by manipulating amino acid transporters presents a highly
promising strategy for increasing crop yield and NUE.

6. Concluding Remarks
Dedicated transport proteins are required to mediate intracellular and intercellular translocation,
and long-distance transport of amino acids within plants. With the completion of genome sequencing
of different plant species, a large number of amino acid transporters have been annotated in plants.
Recently, considerable progress has been made in functionally identifying plant amino acid transporters.
Due to their broad expression pattern and substrate specificity, these transporters may have more than
one physiological function in planta, which requires further exploration. In addition, the functional
characterization of plant amino acid transporters is mainly performed in the model plant Arabidopsis,
while research on their counterparts in major crops is still scarce, which also needs to be investigated
in the future. Furthermore, genetic manipulation of some amino acid transporters can enhance
plant biomass, seed yield and/or quality, as well as NUE, which offers a promising strategy for
crop improvement.

Author Contributions: G.Y. and J.X. wrote the manuscript; Q.W. made the table and revised the manuscript; H.H.
draw the figures. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by National Natural Science Foundation of China (31760065 to G.Y.), Guangxi
Natural Science Foundation (2016GXNSFFA380013 to J.X.) and Guangxi innovation-driven development special
funding project (Guike-AA17204070 to J.X.).
Conflicts of Interest: The authors declare no conflict of interest.

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